22种呼吸道病原体核酸多重联检试剂盒检测效能评估

罗琴 谢会 罗明 李爱华 黄琪 龚成 王雪 李茂中

罗琴, 谢会, 罗明, 李爱华, 黄琪, 龚成, 王雪, 李茂中. 22种呼吸道病原体核酸多重联检试剂盒检测效能评估[J]. 疾病监测.
引用本文: 罗琴, 谢会, 罗明, 李爱华, 黄琪, 龚成, 王雪, 李茂中. 22种呼吸道病原体核酸多重联检试剂盒检测效能评估[J]. 疾病监测.
Luo Qin, Xie Hui, Luo Ming, Li Aihua, Huang Qi, Gong Cheng, Wang Xue, Li Maozhong. Evaluation of multiplex combined real-time PCR detection kit for nucleic acid detection of 22 respiratory pathogens[J]. Disease Surveillance.
Citation: Luo Qin, Xie Hui, Luo Ming, Li Aihua, Huang Qi, Gong Cheng, Wang Xue, Li Maozhong. Evaluation of multiplex combined real-time PCR detection kit for nucleic acid detection of 22 respiratory pathogens[J]. Disease Surveillance.

22种呼吸道病原体核酸多重联检试剂盒检测效能评估

基金项目: 首都卫生发展科研专项(No. 2020-4-3014);科技部传染病重大专项(No. 2017ZX10103004-002);中共北京市委组织部(No. 2018000021469G299)
详细信息
    作者简介:

    罗琴,女,宁夏回族自治区同心县人,在读硕士研究生,研究方向为病原微生物检测,Email:luoluo_never123@163.com

    通讯作者:

    李茂中,Tel:010-64407277,Email:maozhonglee@163.com

  • 中图分类号: R211;R51

Evaluation of multiplex combined real-time PCR detection kit for nucleic acid detection of 22 respiratory pathogens

Funds: This study was supported by the Capital’s Funds for Health Improvement and Research (No.2020-4-3014), Major Project of Infectious Diseases of the Ministry of Science and Technology (No.2017ZX10103004-002), Organization Department of CPC Beijing Municipal Committee (No.2018000021469G299)
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  • 摘要:   目的   通过对1种新型呼吸道病原体核酸多重联检试剂盒检测效能评价,为新型冠状病毒(SARS-CoV-2)、流感病毒等叠加流行的大规模检测提供适宜技术方法。  方法   收集北京地区急性呼吸道感染病例阳性标本252份,采用4种核酸检测试剂盒(3种核酸多重检测试剂盒A、B、C及1种SARS-CoV-2检测试剂盒D)分别进行荧光聚合酶链反应(PCR)检测,评价其与临床标本检测结果的一致性。选取3份呼吸道病原体标本核酸,使用无核糖核酸(RNA)酶水10倍梯度稀释,并选取1份SARS-CoV-2阳性标本核酸稀释为10−1 ~ 10−6,评价A试剂盒的检出能力及批内重复性。  结果   A、B、C试剂盒对甲型流感病毒、鼻病毒、腺病毒、肠道病毒、人偏肺病毒、副流感病毒3型、副流感病毒4型、冠状病毒NL63的检出率均为100%,Kappa值均为1,结果完全一致。对乙型流感病毒、呼吸道合胞病毒、副流感病毒1型、副流感病毒2型、冠状病毒OC43、冠状病毒229E、冠状病毒HKU1及肺炎支原体的Kappa值范围为0.91 ~ 1。3种核酸联检试剂盒对呼吸道病原体的最低检出限相当。A与D试剂盒对SARS-CoV-2的阴阳性符合率均为100%,批内重复性显示在稀释度10−1 ~ 10−3范围内,2种试剂各重复检测5次CV值均 < 5%。在稀释度10−4 ~ 10−5范围内,A试剂对ORF1ab基因检出率为40%,N基因检出率为80%,D试剂对ORF1ab基因检出率为30%,N基因检出率为20%。  结论   A试剂盒与其他3种试剂盒对病毒载量较高的阳性标本检测的准确度无明显差异,检测性能相似;本研究为大规模核酸检测提供了检测试剂盒的多种选择。
  • 图  1  ORF1ab基因与N基因扩增的Ct值分布图

    注:A. A和D 2种试剂对11份阳性标本ORF1ab基因与N基因扩增的Ct值分布图,B. A和D 2种试剂对11份阳性标本ORF1ab基因与N基因检测结果。红色. ORF1ab基因,绿色. N基因,○.和创3.0,△. 伯杰

    Figure  1.  The Ct value of ORF1ab gene and N gene

    图  2  2种试剂盒靶基因的检出能力图

    Figure  2.  The detection capability of two kits

    表  1  A、B、C 3种呼吸道病原核酸多重联检试剂盒标本检测符合率

    Table  1.   Consistency of test results of kit A,B and C

    病原名称检出样本数/
    总样本数
    总符合率(%)Kappa
    阳性
    标本数
    A
    试剂
    B
    试剂
    C
    试剂
    A和
    B试剂
    A和
    C试剂
    A和
    B试剂
    A和
    C试剂
    FluA3131313110010011
    FluB10109999.5999.590.950.95
    H1N11212121210010011
    RHV1818181810010011
    ADV2020202010010011
    EV1818181810010011
    RSV1919171999.171000.941
    HMPV1111111110010011
    H3N21919191910010011
    PIV11110101110099.5910.95
    PIV2656599.591000.911
    PIV31919191910010011
    PIV41313131310010011
    HCoV- OC431715/17/99.17/0.93
    HCoV- NL6388/8/100/1
    HCoV- 229E1615/16/99.59/0.97
    HCoV- HKU11110/11/99.59/0.95
    MP1515151410099.5910.96
      注:FluA. 甲型流感;FluB. 乙型流感;RHV. 鼻病毒;ADV. 腺病毒;EV. 肠道病毒;RSV. 呼吸道合胞病毒;HMPV. 人偏肺病毒;PIV1. 副流感1型;PIV2. 副流感2型;PIV3. 副流感3型;PIV4. 副流感4型;HCoV. 冠状病毒;MP. 肺炎支原体。A、B、C试剂分别代表和创3.0、和创2.0及硕世,其中EV和MP组分不在和创2.0中,而为同一厂家同一批次单独的试剂盒,评价中将2.0单重试剂盒检测的EV与MP结果归为和创2.0。由于和创2.0试剂盒未区分4种常见冠状病毒,因此不计入结果,用“/”表示。共241份标本,其中FluA为甲型H1N1与甲型H3N2共同检测阳性标本,HMPV及PIV1合并感染2例RSV
    下载: 导出CSV

    表  2  4种国产呼吸道病原核酸检测试剂盒的基本信息表

    Table  2.   Information about 4 domestic kits for respiratory tract pathogen nucelic acid detection

    试剂盒组分检测病原内标标本类型检出限
    拷贝/mL
    模板/总体积(ul)循环数/Ct操作步骤
    AA
    B
    C
    D
    E
    F
    FluA,FluB,ORF1ab,N
    H1N1,RHV,ADV
    EV,RSV,HMPV,H3N2
    PIV1,PIV2,PIV3,PIV4
    OC43,229E,HKU1,NL63
    MP,CP,FluB-V,FluB-Y
    咽拭子、痰液、气管抽吸物、肺泡灌洗液、病毒培养物1 0002/2040/35
    1.A-F反应液
    2.酶
    BI
    II
    III
    IV
    PIV2,BOC,RHV
    NL63+OC43,H1N1,HMPV,ADV
    229E+HKU1,FluA,RAV,H3N2
    PIV1,PIV3,PIV4,FluB
    咽拭子、鼻咽拭子、痰液1 0002/2040/351.反应液
    2.酶
    CA
    B
    C
    D
    E
    F
    G
    H
    FluA,FluB,H1N1,H3N2
    PIV1,PIV2,PIV3,PIV4
    RHV,HMPV,RSV
    BOC,ADV,EV
    229E,MERS,SARS
    OC43,HKU1,NL63
    MP,CP. SP,Hib
    KPn,BP,Lcn
    咽拭子等标本1 0005/2540/351.RT-PCR反应液
    2.A-H反应液
    3.酶
    4.去RNA酶水
    DORF1ab,N咽拭子等标本1 0005/2540/381.核酸扩增反应液
    2.酶
    3.ORF1ab/N反应液
      注:FluA. 甲型流感;FluB. 乙型流感;RHV. 鼻病毒;ADV. 腺病毒;EV. 肠道病毒;RSV. 呼吸道合胞病毒;HMPV. 人偏肺病毒;PIV1. 副流感1型;PIV2. 副流感2型;PIV3. 副流感3型;PIV4. 副流感4型;HCoV. 冠状病毒;MP. 肺炎支原体。A. 呼吸道病原体核酸多重联检试剂盒3.0、B. 呼吸道病原体核酸多重联检试剂盒2.0、C. 呼吸道27种病原体核酸检测试剂盒、D. 新型冠状病毒2019-nCoV核酸检测试剂盒。其中A-F及I-VI代表不同的组分
    下载: 导出CSV

    表  3  A、B、C 3种核酸联检试剂盒对常见呼吸道病原体的最低检出限

    Table  3.   Comparison of the detection capability of kits A to C

    病原名称第1份标本检出限第2份标本检出限第3份标本检出限
    A试剂C试剂B试剂A试剂C试剂B试剂A试剂C试剂B试剂
    FluA10−310−310−210−310−310−210−310−310−3
    FluB10−310−310−310−210−210−210−310−310−3
    H1N110−310−310−310−310−310−310−310−310−3
    RHV10−310−310−210−210−410−310−310−310−3
    ADV10−410−410−310−210−310−210−310−310−3
    EV10−310−3/10−310−2/10−110−2/
    RSV10−410−410−410−410−410−410−310−310−3
    HMPV10−210−310−310−310−310−210−310−210−2
    H3N210−410−410−210−310−410−210−310−310−2
    PIV110−310−310−310−310−310−210−210−310−2
    PIV210−410−4/10−410−4/10−410−4/
    PIV310−210−210−210−310−310−310−310−310−2
    PIV410−310−310−310−410−410−310−410−410−3
    HCoV-OC4310−310−4/10−310−4/10−310−3/
    HCoV-NL6310−310−3/10−310−3/10−410−4/
    HCoV-229E10−410−4/10−310−4/10−310−3/
    HCoV-HKU110−310−4/10−310−3/10−410−4/
    MP10−310−3/10−310−3/10−310−2/
      注:FluA. 甲型流感;FluB,乙型流感;RHV. 鼻病毒;ADV. 腺病毒;EV. 肠道病毒;RSV. 呼吸道合胞病毒;HMPV.人偏肺病毒;PIV1. 副流感1型;PIV2. 副流感2型;PIV3. 副流感3型;PIV4. 副流感4型;HCoV. 冠状病毒;MP. 肺炎支原体。A、B、C分别代表和创3.0、和创2.0和硕世等3种试剂盒,/. 未做稀释度检测
    下载: 导出CSV

    表  4  A与D试剂盒对不同稀释度阳性标本的批内重复性结果

    Table  4.   The result of intra-batch repeatability with Kit A and D

    不同稀释度靶基因检测A试剂盒D试剂盒
    均值±标准差(拷贝/mL)CV(%)均值±标准差(拷贝/mL)CV(%)
    10−1
    ORF1ab22.10±0.221.0025.40±0.552.17
    N20.02±0.040.2026.40±0.893.37
    10−2
    ORF1ab25.30±0.451.7829.06±0.130.45
    N23.80±0.271.1330.20±0.451.49
    10−3
    ORF1ab28.08±0.180.6431.60±0.892.82
    N27.04±0.092.50 33.20±0.832.50
    10−4
    ORF1ab
    N30.20±0.451.49
      注:A. 多重核酸联检试剂盒(和创3.0),D. SARS-CoV-2核酸检测试剂盒(硕世),其中“-”代表未完全检出
    下载: 导出CSV
  • [1] Bryce J, Boschi-Pinto C, Shibuya K, et al. WHO estimates of the causes of death in children[J]. Lancet, 2005, 365(9465): 1147–1152. DOI: 10.1016/S0140−6736(05)71877−8.
    [2] Zhang NR, Wang LL, Deng XQ, et al. Recent advances in the detection of respiratory virus infection in humans[J]. J Med Virol, 2020, 92(4): 408–417. DOI:  10.1002/jmv.25674.
    [3] Wen SH, Lv FF, Chen XF, et al. Application of a nucleic acid-based multiplex kit to identify viral and atypical bacterial aetiology of lower respiratory tract infection in hospitalized children[J]. J Med Microbiol, 2019, 68(8): 1211–1218. DOI:  10.1099/jmm.0.001006.
    [4] Malhotra B, Swamy MA, Janardhan Reddy PV, et al. Evaluation of custom multiplex real-time RT-PCR in comparison to fast-track diagnostics respiratory 21 pathogens kit for detection of multiple respiratory viruses[J]. Virol J, 2016, 13: 91. DOI: 10.1186/s12985−016−0549−8.
    [5] 邸红芹, 杨永辉, 康丽菲, 等. 多种呼吸道病原体的多重实时荧光定量PCR检测方法的建立及应用[J]. 河北医科大学学报,2016,37(11):1302–1306. DOI:10.3969/j.issn.1007−3205.2016.11.016.

    Di HQ, Yang YH, Kang LF, et al. Establishment and application of multiplex real-time fluorescence quantitative PCR assay for detecting various respiratory pathogens[J]. J Hebei Med Univ, 2016, 37(11): 1302–1306. DOI: 10.3969/j.issn.1007−3205.2016.11.016.
    [6] Loeffelholz MJ, Tang YW. Laboratory diagnosis of emerging human coronavirus infections-the state of the art[J]. Emerg Microbes Infect, 2020, 9(1): 747–756. DOI:  10.1080/22221751.2020.1745095.
    [7] Chan JFW, Yip CCY, To KKW, et al. Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-PCR assay validated in vitro and with clinical specimens[J]. J Clin Microbiol, 2020, 58(5): e00310–20. DOI: 10.1128/JCM.00310−20.
    [8] 国家卫生健康委员会卫生应急办公室. 截至1月26日24时新型冠状病毒感染的肺炎疫情最新情况[EB/OL]. (2020-01-27)[2021-05-10]. http://www.nhc.gov.cn/xcs/yqtb/202001/3882fdcdbfdc4b4fa4e3a829b62d518e.shtml.

    Health Emergency Office of National Health Commission. Update on the outbreak of pneumonia infected by novel coronavirus as of 24: 00 January 26[EB/OL]. (2020-01-27) [2021-05-10].. http://www.nhc.gov.cn/xcs/yqtb/202001/3882fdcdbfdc4b4fa4e3a829b62d518e.shtml.
    [9] 熊丹, 阚丽娟, 王萌萌, 等. 七种国产新型冠状病毒核酸检测试剂盒的一致性和检出能力评价研究[J]. 中华检验医学杂志,2020,43(8):787–793. DOI:10.3760/cma.j.cn114452−20200227−00133.

    Xiong D, Kan LJ, Wang MM, et al. Evaluation of the consistency and detection capability of seven domestic 2019-nCoV nucleic acid detection kits[J]. Chin J Lab Med, 2020, 43(8): 787–793. DOI: 10.3760/cma.j.cn114452−20200227−00133.
    [10] Zhou YY, Pei FY, Ji MY, et al. Sensitivity evaluation of 2019 novel coronavirus (SARS-CoV-2) RT-PCR detection kits and strategy to reduce false negative[J]. PLoS One, 2020, 15(11): e0241469. DOI:  10.1371/journal.pone.0241469.
    [11] Liu YW, Wang YY, Wang XM, et al. Development of two TaqMan real-time reverse transcription-PCR assays for the detection of severe acute respiratory syndrome coronavirus-2[J]. Biosaf Health, 2020, 2(4): 232–237. DOI:  10.1016/j.bsheal.2020.07.009.
    [12] Chung HY, Jian MJ, Chang CK, et al. Novel dual multiplex real-time RT-PCR assays for the rapid detection of SARS-CoV-2, influenza A/B, and respiratory syncytial virus using the BD MAX open system[J]. Emerg Microbes Infect, 2021, 10(1): 161–166. DOI:  10.1080/22221751.2021.1873073.
    [13] Wu XJ, Cai Y, Huang X, et al. Co-infection with SARS-CoV-2 and influenza a virus in patient with pneumonia, China[J]. Emerg Infect Dis, 2020, 26(6): 1324–1326. DOI:  10.3201/eid2606.200299.
    [14] Kondo Y, Miyazaki S, Yamashita R, et al. Coinfection with SARS-CoV-2 and influenza a virus[J]. BMJ Case Rep, 2020, 13(7): e236812. DOI: 10.1136/bcr−2020−236812.
    [15] 陈涛, 杨静, 汪立杰, 等. 2015年中国大陆流行性感冒流行特征分析[J]. 热带病与寄生虫学,2016,14(1):3–5. DOI:10.3969/j.issn.1672−2302.2016.01.002.

    Chen T, Yang J, Wang LJ, et al. Analysis on the epidemiological characteristics of influenza in mainland China in 2015[J]. J Trop Dis Parasitol, 2016, 14(1): 3–5. DOI: 10.3969/j.issn.1672−2302.2016.01.002.
    [16] Radko S, Ian Stuart J, Zahariadis G. Evaluation of three commercial multiplex assays for the detection of respiratory viral infections[J]. J Virol Methods, 2017, 248: 39–43. DOI:  10.1016/j.jviromet.2017.06.006.
    [17] Done M, Luo M, Li AH, et al. Changes in the pathogenic spectrum of acute respiratory tract infections during the COVID-19 epidemic in Beijing, China: a large-scale active surveillance study[J]. J Infect, 2021. DOI:  10.1016/j.jinf.2021.08.013.
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